The present invention relates essentially to a collagen product containing collagen of marine origin with a low odor and preferably with improved mechanical properties, to its use in the form of cosmetic or pharmaceutical compositions or products and to the carrying out of tests in vitro, for example in the context of reconstructed tissues. The invention further relates to a process for the preparation of this collagen product containing collagen of marine origin with a low odor.
In an earlier patent application published under No. WO 93/01241, the present Applicant described the use of the unpigmented skin of fish, particularly flatfish, as a novel industrial source of collagen, the process for its extraction and the collagen and biomaterials obtained by said process.
Unpigmented marine collagen, as obtained by the Applicant""s previous invention, made it possible to obtain collagen industrially at an acceptable cost by virtue of the large quantities of skins available in industry, particularly skins originating from flatfish.
However, the present inventors set themselves the new technical problem of the odor generated by collagen derived from fish, in solid form; this problem did not arise within the framework of said earlier patent application insofar as the main application concerned its use as one of the components of cosmetic or pharmaceutical compositions in which the other components, present in very large majority, masked the odor by dilution.
The present inventors also set themselves the new technical problem of the odor normally generated by collagen products containing collagen of marine origin obtained from marine animals such as fish, jellyfish, molluscs and shellfish, irrespective of the form of the collagen, including hydrolyzates thereof. In fact, it has also been possible to observe that not only collagens, irrespective of their forms, whether native or not, in the form of atelocollagen, insoluble collagen, particularly collagen fibers, soluble collagen, particularly acid-soluble collagen, or hydrolyzates thereof, have a troublesome odor.
The present inventors have also been able to observe that, especially in the context of the formation of sponges, membranes or masks of collagen products in which the concentration of collagen(s) or hydrolyzates thereof is relatively high, the odor problem becomes troublesome and severely restricts its use, particularly in the field of cosmetics or pharmaceuticals and especially as a biomaterial for topical application, particularly as a mask.
By way of a new technical problem, the present inventors also set themselves the problem of the insufficiency of the mechanical properties of solid biomaterials prepared from a collagen product of marine origin.
Thus the object of the present invention is to solve both the above-mentioned technical problems individually and preferably simultaneously, and particularly preferably without affecting or degrading the collagen(s), making it possible especially to preserve the native character of this collagen or these collagens.
According to the present invention, a solution to both these problems has unexpectedly been found which also makes it possible to obtain the collagen product with satisfactory mechanical properties, especially by virtue of the fact that, if desired, the invention makes it possible to preserve the native character of the collagen.
Thus, according to a first feature, the present invention provides, by way of a novel product, a collagen product containing collagen of marine origin obtained from marine animals such as fish, jellyfish, molluscs and shellfish, which product comprises one or more collagens or derivatives thereof, including hydrolyzates, with a low odor, at least part of said collagen or said derivatives being of marine origin and having been subjected to a deodorization treatment at any stage of the manufacture of the collagen product.
The expression xe2x80x9ccollagen productxe2x80x9d is understood within the framework of the present invention as meaning any product which contains at least one collagen, and any derived product, including hydrolyzates. xe2x80x9cCollagenxe2x80x9d is understood as meaning any form of collagen, whether native or not, atelocollagen, insoluble collagen, particularly collagen fibers, soluble collagen, particularly acid-soluble collagen, and any derived product, including hydrolyzates. These hydrolyzates can be obtained chemically or enzymatically in a manner well known to those skilled in the art.
Moreover, the collagen or derivatives thereof can be in totally minor or major proportions, irrespective of its form.
Thus the collagen product according to the invention can comprise from 1 to 100% by weight of collagen(s) and derivatives thereof, including hydrolyzates thereof, preferably from 10 to 100% by weight of these products.
Within the framework of the invention, the above-mentioned deodorization treatment can be carried out initially on the collagen(s), derivatives thereof or hydrolyzates thereof, by themselves or in isolation, or on the final collagen product.
Advantageously this low-odor collagen product is obtained by means of a treatment with at least one active substance having a deodorizing effect.
Said active substance is advantageously an oxidizing substance. As the oxidizing substance it is currently preferable to use at least one sulfite, particularly a metabisulfite, or a salt of said sulfite or metabisulfite, and/or hydrogen peroxide or ozone.
An alkali metal or alkaline earth metal, preferably an alkali metal such as sodium or potassium, may be used in particular as the salt of a sulfite. It is also possible to use a partial salt of a sulfite, like metabisulfite or hydrogensulfite, with an alkali metal or alkaline earth metal as defined above.
The proportion of oxidizing substances, particularly sulfite, varies between very wide limits and will generally be between 0.001% and 20% by weight, advantageously between 0.01% and 10% by weight. A proportion which ensures an industrially effective deodorization treatment may be of the order of about 5% by weight, based on the composition containing the marine collagen to be deodorized.
In one particular embodiment of the invention, this deodorization treatment may be carried out on the collagen-containing parts of marine animals, particularly finely divided fish skins, for example fish skins which have been ground.
In another advantageous embodiment of the invention, the deodorization treatment may be carried out on collagen fibers which have been precipitated from a collagen solution obtained from fish skins in at least a first extraction step, or these two treatments may be combined.
In another advantageous embodiment, the deodorization treatment is carried out on a collagen gel or a solution of collagen hydrolyzate brought into contact with ozone, particularly at an ozone flow rate of between 25 and 50 l/hour per kg of gel, the ozone content being between 20 and 50 mg/l. This deodorization with ozone may be effected by bubbling this gas into the collagen gel or the solution of collagen hydrolyzate.
According to yet another advantageous characteristic of the invention, which is actually preferred, this collagen product has improved mechanical properties and has undergone a crosslinking treatment which initially cannot be a chemical crosslinking; this is because the latter takes place in a liquid or aqueous medium on a solid sponge of very low mechanical strength and generally culminates in a disaggregation of the collagen sponge. Thus, within the framework of the invention, it is preferable to carry out a non-chemical crosslinking, preferably a physical crosslinking and particularly preferably a crosslinking by thermal dehydration, abbreviated to TDH. In this context it is preferable to carry out a thermal dehydration under vacuum at a temperature above 60xc2x0 C., advantageously of between about 110 and about 130xc2x0 C. and particularly of the order of 110xc2x0 C.
After a first non-chemical crosslinking has been carried out, preferably by TDH, it is then possible to strengthen the crosslinking further by performing a chemical crosslinking because, at this point, the mechanical strength of the collagen is sufficiently high to allow a chemical crosslinking. Chemical crosslinking agents are known to those skilled in the art and an example which may be mentioned is the crosslinking with azide described in earlier patent application Ser. No. 5,264,551 in the name of the Applicant.
The source of marine collagen used is preferably fish skins, it being possible to use any fish skins in this context. Advantageously, however, it is preferable to use a fish skin which is collected industrially because it is generally cut away from the fresh fish directly at the fishing site and is advantageously frozen immediately after removal.
It is generally flatfish which are subjected to a skinning step to extract the fillets, so the skins of flatfish are a practical source of fish skins.
For certain applications it may be advantageous to obtain unpigmented collagen in the manner described in previous patent application WO 93/01241 or U.S. Pat. No. 5,420,248 in the name of the Applicant, so an unpigmented fish skin may be used in certain types of application as the source for preparing the collagen.
Similarly it is also possible to prepare collagen films by drying a collagen gel in a stream of gas, particularly in air, and optionally subjecting it to a physical crosslinking step, particularly by thermal dehydration, which can be followed by a chemical crosslinking step.
According to a second feature, the present invention also covers a sponge, membrane or mask comprising marine collagen preferably obtained from fish skins, wherein the collagen has been subjected to a deodorization treatment, advantageously as defined above, during its manufacturing process. According to an advantageous characteristic, the sponge, membrane or mask comprises marine collagen which has also been subjected to at least one physical crosslinking as described above, particularly a crosslinking by TDH, by itself or in combination with a subsequent chemical crosslinking, in order to improve its mechanical properties.
According to a third feature, the invention further relates to a process for the manufacture of a collagen product containing collagen of marine origin, which comprises carrying out a deodorization treatment on the collagen itself or on the collagen product, this deodorization treatment preferably being carried out with at least one active substance having a deodorizing effect.
Other advantageous characteristics of the treatment process are clearly apparent to those skilled in the art from the above description relating to the collagen product itself, and from the Examples below, which are given simply by way of illustration but form an integral part of the invention. In the Examples, any characteristic which appears to be novel compared with any state of the art forms an integral part of the present invention in most instances. In addition, all the percentages are given by weight, unless indicated otherwise.
Within the framework of the invention, the collagen product can contain collagen which has been subjected to a deodorization treatment, optionally mixed with untreated collagen or collagen from a source other than a marine source.
Furthermore, among marine animals, fish are currently the preferred source of collagen of marine origin, particularly fish skins.
According to a fourth feature, the invention further relates to cosmetic or pharmaceutical products, especially biomaterials, which are partially or totally produced from the marine collagen product with a low odor and preferably with improved mechanical properties, and especially to sponges, membranes or masks of a marine collagen product, as defined above.
As far as cosmetic products are concerned, the invention is particularly advantageous in the context of collagen-based cosmetic masks. In fact, those skilled in the art are well aware that collagen derived from mammals is to be avoided insofar as this collagen might carry a risk of contamination, and also for ecological reasons. In addition, for reasons of image, the majority of cosmetics companies are increasingly using fewer ingredients originating from mammals. Marine collagen, on the other hand, has so far been unable to find an application because of its strong and unpleasant odor and its low mechanical strength.
The invention therefore makes it possible to provide a satisfactory solution to these two problems which the present inventors were the first to set themselves. In the case of the invention, insofar as it is desirable or necessary for the collagen to have very good mechanical properties, the totally native character of the collagen is very important. A collagen of native character is understood as meaning a collagen which has preserved its helical structure as well as its telopeptides, which carry a large or even major proportion of the collagen""s crosslinkages.
Within the framework of the invention, at least one or more cosmetic or pharmaceutical active principles may be incorporated into or added to the collagen, depending on the envisaged application of the collagen, as can easily be understood by those skilled in the art.
By virtue of the invention, it is now possible to use marine collagen in cosmetic or pharmaceutical products, particularly in biomaterials and especially masks, which consist of very thin collagen sponges a few millimeters thick, intended for application to the skin and more particularly to the face. The purpose of these masks is to smooth and whiten the skin. They also give a substantial sensation of well-being. They can serve as a carrier for active ingredients, which will then be released gradually onto the surface of the skin. In this case the active substance can either be introduced into the whole volume of the sponge during manufacture or be applied to the surface of the mask when the latter is in contact with the skin.
In the pharmaceutical field, collagen can be used in the form of sponges in numerous applications: hemostatic sponges, healing dressings and cell culture supports intended for the engineering of tissues, especially reconstructed skin and cartilage. For these applications it is highly desirable to use a collagen which does not originate from a mammal; the present invention makes it possible to meet this requirement.
It should be noted that fish skin is a preferred raw material. It actually has several merits: firstly, it is available in large quantities, particularly in the case of fish consumed as fillets, making it relatively inexpensive; secondly, it is removed industrially under conditions of high cleanliness; finally, it is frozen very rapidly after removal, ensuring that the collagen preserves its native character. The native character is also preserved by the process according to the present invention.